Neuroinflammatory responses may be dependent on the initiation of innate immune responses triggered by the stimulation of intrinsic brain cells by pathogen-associated molecular patterns (PAMPs), repeated structural motifs generated by microbes that are not normally found in the host or by debris from apoptotic or necrotic cells following injury. One group of receptors that recognize viral PAMPs is the toll-like receptor (TLR) 9 family, which is composed of TLR7, TLR8 and TLR9. These receptors form a closely related family that are highly homologous, confined to the endosomal membranes and recognize nucleic acids that are encountered in these compartments. TLR7 and TLR8 recognize single stranded RNA (ssRNA) from a number of virus families, while TLR9 recognizes unmethylated DNA containing CpG motifs (CpG DNA). There are several recent studies demonstrating that TLR9 family members play an important role in neuropathogenesis, including our study demonstrating that TLR7 plays a role in retrovirus-induced innate immune responses in the central nervous system (CNS). Additionally, TLR7 and TLR9 agonists are being studied as potential therapeutics for multiple diseases in the brain, ranging from virus infections to the treatment of brain tumors. However, there is a lack of basic understanding of which cell types in the brain respond to stimulation of TLRs, as well as the pathways of neuroinflammation, neuroprotection and/or neuronal damage induced when these TLRs are activated. Understanding the similarities and differences of TLR7, TLR8 and TLR9-induced cell activation in the brain is important for understanding viral pathogenesis as well as potential use of TLR agonists in the treatment of neurological diseases. Our laboratory has focused on understanding the response of intrinsic brain cells following TLR activation and determining the downstream effects of TLR activation on neuroinflammation and neuropathogenesis. In FY2010, we analyzed the response of astrocytes, microglia and neurons to TLR7 and/or TLR9 activation. We found that glial cells responded similarly to TLR7 or TLR9 activation, although the cytokine profiles elicited by TLR stimulation of these cells differed. Importantly, we discovered cross-regulation between TLR7 and TLR9 in inducing neuroinflammatory responses. TLR7 ligands and well as TLR7, itself, were shown to inhibit TLR9-mediated responses in glial cells. This result demonstrates a new complexity in the regulation of inflammation in the CNS.